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Abstract
A mixed bacterial culture consisted of Staphylococcus sp., Bacillus circulans-I and -II has been enriched from contaminated soil collected from
the vicinity of an endosulfan processing industry. The degradation of endosulfan by mixed bacterial culture was studied in aerobic and facultative
anaerobic conditions via batch experiments with an initial endosulfan concentration of 50 mg/L. After 3 weeks of incubation, mixed bacterial culture
was able to degrade 71.58 0.2% and 75.88 0.2% of endosulfan in aerobic and facultative anaerobic conditions, respectively. The addition of
external carbon (dextrose) increased the endosulfan degradation in both the conditions. The optimal dextrose concentration and inoculum size
was estimated as 1 g/L and 75 mg/L, respectively. The pH of the system has significant effect on endosulfan degradation. The degradation of
alpha endosulfan was more compared to beta endosulfan in all the experiments. Endosulfan biodegradation in soil was evaluated by miniature
and bench scale soil reactors. The soils used for the biodegradation experiments were identified as clayey soil (CL, lean clay with sand), red soil
(GM, silty gravel with sand), sandy soil (SM, silty sand with gravel) and composted soil (PT, peat) as per ASTM (American society for testing and
materials) standards. Endosulfan degradation efficiency in miniature soil reactors were in the order of sandy soil followed by red soil, composted
soil and clayey soil in both aerobic and anaerobic conditions. In bench scale soil reactors, endosulfan degradation was observed more in the bottom
layers. After 4 weeks, maximum endosulfan degradation efficiency of 95.48 0.17% was observed in red soil reactor where as in composted soil-I
(moisture 38 1%) and composted soil-II (moisture 45 1%) it was 96.03 0.23% and 94.84 0.19%, respectively. The high moisture content
in compost soil reactor-II increased the endosulfan concentration in the leachate. Known intermediate metabolites of endosulfan were absent in all
the above degradation studies.
2005 Elsevier B.V. All rights reserved.
Keywords: Endosulfan; Mixed culture; Biodegradation; Staphylococcus sp.; Bacillus circulans
1. Introduction
Endosulfan, a sulphurous acid ester of a chlorinated cyclic
diol [1] is a mixture of two stereo isomers alpha and beta endosulfan in a ratio of 7:3 (Fig. 1) and registered with several trade
marks, Thiodan, Cyclodan, Thimol, Thiofar and Malix. It is
extremely toxic to fish and aquatic invertebrates and has been
implicated in mammalian toxicity [2], genotoxicity [3], and neurotoxicity [4]. It enters the air, water, and soil environments
during its use, and manufacture. Endosulfan is highly insolu-
Corresponding author. Tel.: +91 44 2257 4274; fax: +91 44 2257 4252.
E-mail address: ligy@iitm.ac.in (L. Philip).
0304-3894/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jhazmat.2005.12.023
355
(CL, lean clay with sand), red soil (GM, silty gravel with
sand), composted soil (PT, peat) and sandy soil (SM, silty
sand with gravel) [19]. The soils were sieved through IS
sieve No. 10 (2 mm aperture as per IS 2720 (Part 4), 1987).
The fraction passing through the sieve was collected and
preserved in air tight plastic containers for biodegradation
studies.
3. Analytical techniques
Endosulfan, its isomers and degradation products, i.e. endosulfan sulfate, endosulfan ether and endosulfan lactone were
analyzed by Perkin-Elmer Clarus 500 gas chromatograph with
electron capture detection (GC/ECD). Under these conditions
the retention time for alpha endosulfan, beta endosulfan, endosulfan sulfate, endosulfan ether and endosulfan lactone was
6.31, 9.36, 11.95, 3.72 and 6.42 min, respectively, and the chromatogram is shown in Fig. 2(a) [19].
Bacterial cells in the systems were measured using a UV
digital spectrometer (Techcomp, Hong Kong) in fixed-point
measurement at 550 nm [20].
Most common Indian soils were selected for the biodegradation experiments and they were classified as clayey soil
356
Fig. 2. (a) Chromatogram of endosulfan and its metabolites at standard operating condition. (b) Chromatogram of reactor effluent extracts during biodegradation at
5, 7, 14 and 21 days at standard operating condition.
NB to receive a final endosulfan concentration of 50 ppm. Dextrose was supplied as a supplementary carbon at concentrations
of 1, 2, 3 and 4 g/L and the pH of the medium was maintained
at 7. Predetermined concentration of bacterial cells corresponds
to OD550 0.15 (75 mg/L) were inoculated and the flasks were
kept in an orbital shaker at 28 2 C at 150 rpm. The studies
were conducted in both aerobic and facultative anaerobic conditions for 21 days. Nitrogen gas was purged into anaerobic
flasks and immediately sealed with air tight septum to maintain the anaerobic condition. At various time intervals (0, 2, 5,
7, 10, 14 and 21 days) samples were withdrawn from the conical flasks and analyzed for residual endosulfan concentration
using GC/ECD. After collecting the samples from anaerobic
flasks, nitrogen gas was purged again and sealed with air tight
septum.
Fig. 3. (a) Schematic diagram of miniature soil reactor; (b) bench scale soil
reactor.
357
358
0
1
2
3
4
71.29
81.66
81.71
82.94
84.91
0.09
0.15
0.08
0.11
0.11
Anaerobic system
Beta
endosulfan
72.27
78.20
78.40
77.80
78.33
0.02
0.03
0.03
0.02
0.05
Alpha
endosulfan
76.40
85.17
85.43
85.97
85.77
0.13
0.13
0.11
0.15
0.13
Beta
endosulfan
74.67
85.00
85.13
86.27
86.46
Table 2
Growth of mixed and pure bacterial cultures during degradation of endosulfan in the presence and absence of dextrose
0
2
5
7
10
14
18
21
28
Aerobic
Aerobic
Aerobic
Anaerobic
Aerobic
75
100
175
210
245
270
75
120
195
240
265
275
75
115
190
240
270
280
75
120
210
255
295
320
345
355
350
4
4
3
6
10
8
8
7
6
Anaerobic
75
90
155
210
245
275
300
320
325
3
5
7
7
7
10
5
7
6
75
140
245
280
315
335
360
375
365
4
8
5
5
6
9
9
6
6
0.03
0.05
0.03
0.05
0.04
Fig. 4. (a) Degradation of endosulfan at various supplementary carbon concentrations in aerobic system; (b) in facultative anaerobic system.
Time (days)
Anaerobic
75
110
180
220
260
295
320
340
340
6
5
6
8
8
10
7
8
5
4
5
5
4
6
6
3
7
4
9
7
7
5
8
8
11
8
9
359
inoculum size is a rate limiting factor in the degradation of endosulfan in aerobic process compared to anaerobic process. When
the bacterial concentration in the system was 75 mg/L, better
performance of the system was observed in both aerobic and
facultative anaerobic conditions. Hence, it can be concluded that
an inoculum size of 75 mg/L can be used as an optimum dose
for endosulfan degradation experiments.
5.3. Effect of pH
Batch experiments were conducted in aerobic and facultative
anaerobic conditions to study the influence of pH on endosulfan
degradation by the microbial consortium. In the present study,
endosulfan was amended directly into nutrient broth (NB) and
endosulfan degradation experiments were conducted at various
pH, i.e. 4, 6, 7, 8 and 10. These pH values are corresponds
to the initial pH of the NB. Later, the pH of the system was
decreased. At the end of the study, pH of the systems was 4.4,
6.2, 7.1, 7.9 and 8.3, respectively. In aerobic condition, at the
end of 21 days, endosulfan degradation efficiency (at pH 4) was
55.7 0.22%. The increase in pH value to 6 and 7 increased
the degradation efficiency to 71.3 0.19% and 71.58 0.27%,
respectively. Further increase in pH value to 8 reduced the
efficiency marginally (71.28 0.2%) and at pH 10 it reduced
drastically (37.56 0.3%). Decrease in pH to 4 and increase
in pH to 10 reduced the endosulfan degradation efficiency by
22.18 1.4% and 47.53 1.45% (compared to neutral pH) at
the end of 21 days (Fig. 7(a)). Similar trends were observed by
Siddique et al. [17] while studying the endosulfan degradation
by enriched fungal and bacterial strains. This may be due to the
decreased growth of microbes at extreme pH. It is reported that
at high pH, hydrolysis of endosulfan was faster [22,23]. In the
present system, nutrient medium was present along with endosulfan in control reactors. Though the initial pH was increased to
10, the final pH reduced to 8.3 at the end of 21 days, whereas, no
significant change in the pH value (pH 9.9 0.1) was observed
in the blank reactor (operated at pH 10) at the end of 21 days.
360
Fig. 7. (a) Degradation of endosulfan at various pH in aerobic system. (b) Variation of endosulfan metabolites in blank reactor (distilled water) at pH 10. (c)
Degradation of endosulfan at various pH in facultative anaerobic system; (d) in aerobic and facultative anaerobic system at the end of 21 days.
The decrease in pH might have retarded the alkaline hydrolysis. From the control studies (endosulfan amended directly in
NB), it was observed that no intermediate metabolites of endosulfan were detected in any of the system and the abiological
loss of endosulfan in the controlled flasks was negligible. Later,
the control studies were repeated in distilled water (endosulfan
amended in distilled water without NB) where endosulfan diol
and endosulfan ether was observed due to alkaline hydrolysis in
both aerobic and facultative anaerobic conditions. Around 20%
of endosulfan was hydrolyzed to endosulfan diol and endosulfan
ether. This value was converted to parent compound equivalents
and it was around 99%. This emphasize the fact that endosulafan
is not getting removed from the system by volatilization. The
accumulation of endosulfan diol and endosulfan ether, in blank
reactors, operated in aerobic and facultative anaerobic systems
is shown in Fig. 7(b).
In facultative anaerobic process (at pH 4) endosulfan
degradation efficiency was 38.64 0.28% and it was increased
to 55.72 0.21% at pH 6. The reduction in efficiency of the
system due to decrease in pH to 4 and 6 was 47.76 1.3%
361
Fig. 8. (a) Kinetics of endosulfan degradation in miniature soil reactors in aerobic condition; (b) in facultative anaerobic condition. (c) Endosulfan degradation in
miniature soil reactors at the end of 28 days in aerobic condition; (d) in facultative anaerobic condition.
362
Table 3
Endosulfan degradation in bench scale soil reactors for different soils
Type of
soil
Moisture
content (%)
Red soil
CS-Ia
CS-IIa
a
38 1
38 1
45 1
Middle
Bottom
Top
Middle
Bottom
Composted soil.
made to change the soil pH during the bench scale soil reactor
studies.
During the operation, the simulated bioreactor has shown two
distinct zones, i.e. top of the reactor was in aerobic condition,
whereas, bottom layers were in anaerobic condition. The entry of
air to the bottom layers may be restricted due to the compaction
of soil and no attempts were made to supply the air in the bottom zone of the reactors. The loss in moisture in the reactor was
Fig. 9. (a) Alpha endosulfan degradation in bench scale reactors. (b) Beta endosulfan degradation in bench scale reactors.
363
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